Since the 1920's it has been observed that cancer cells (relative to normal cells) demonstrate increased rates of glycolysis and pentose cycle activity as well as slightly decreased rates of respiration but the significance of this to cancer therapy is unclear. Recent studies have shown that glucose deprivation preferentially induces cytotoxicity and oxidative stress in human cancer cells, relative to normal cells [Appendix 1]. Mitochondria have been hypothesized to be the site of prooxidant production during glucose deprivation. If this were generally true, glucose deprivation-induced oxidative stress could represent a defect in tumor cell mitochondrial metabolism amenable to manipulations designed to improve cancer therapy. The current proposal will test the hypothesis that mitochondrial production of reactive oxygen species (ROS; i.e., superoxide and hydrogen peroxide) mediates the increased susceptibility of human cancer cells to glucose deprivation-induced metabolic oxidative stress, relative to normal cells. Specific Aim 1 will determine using electron transport chain blockers (i.e., antimycin A, myxothiazol, and rotenone), if intact human cancer cells (or isolated mitochondria) demonstrate alterations in ROS production by mitochondrial electron transport chain Complexes I, II, and/or III that contribute to increased susceptibility to glucose deprivation-induced oxidative stress, relative to normal cells. Specific Aim 2 will determine if rho(0) cancer cells, deficient in functional mitochondrial electron transport chains demonstrate altered susceptibility to glucose deprivation-induced cytotoxicity and oxidative stress, relative to parental rho(+) cells containing fully functional electron transport chains. Specific Aim 3 will determine using adenoviral vectors as well as stably transfected cell lines, if over expession of antioxidant enzymes that scavenge superoxide and hydrogen peroxide (i.e., catalase, superoxide dismutases) are capable of altering the biological effects of glucose deprivation in cancer cells. Specific Aim 4 will determine if 2-deoxy-d-glucose is capable of mimicking the effects of glucose deprivation seen in Aims 1-3. The long-term goal is to provide a rigorous mechanistic understanding of the differential susceptibility of cancer cells to glucose deprivation-induced oxidative stress for the purpose of developing combined modality cancer therapy based on differences between oxygen metabolism in normal vs. cancer cells.